Cytokinesis is a highly coordinated cellular process achieved by contractile ring formation, upon subsequent contraction of this ring one cell divides to form two cells. Temporal and spatial regulation of the cytokinetic machinery is pivotal to ensure equal partitioning of genomic and cellular materials into two dividing cells. In budding yeast, the cleavage plane is specified early in the cell cycle and cleavage is achieved by a concerted process of membrane constriction by an actomyosin-based contractile ring followed by septum formation. Septins (Cdc3, Cdc10, Cdc11, and Cdc12) are the major structural components of bud-neck filaments and thought to function as a scaffold for the recruitment of cytokinesis machinery to the mother-bud neck. We have found that a novel neck-filament-associated protein, Bni5, plays an important role for the stability of septin ring structures. Loss of bni5 function leads to an enhanced cytokinetic defect with all four septin mutants (cdc3-6, cdc10-1, cdc11-6, and cdc12-6), whereas overexpression of BNI5 suppresses both the cytokinetic and cell growth defects associated with the cdc10-1, cdc11-6, or cdc12-6 mutant. Bni5 protein localizes at the neck filaments in a septin-dependent manner and interacts with Cdc11 and Cdc12 in a yeast-two hybrid system. In addition, the coiled-coil domain present in the C-terminus of Bni5 appears to be critical for localization. Our data suggest that Bni5 may directly interact with septins to provide stability for septin ring structures that is critical for normal cytokinesis.